Over 90% of ethanol biofuel produced in the United States is made from corn starch using Saccharomyces strains to ferment the glucose obtained by hydrolysis of the starch. The United States Environmental Protection Agency has revised the Renewable Fuel Standard (RFS) program as required by the Energy Independence and Security Act of 2007 (EISA). The final rule (RFS2) increases the volume requirements for total renewable fuel to 20.5 billion gallons and for cellulosic biofuel to 3.0 billion gallons by 2015. To meet these mandates, it will be necessary to use cellulosic biomass, an abundant and renewable carbon source, as a feedstock. However, the microbial strains used to ferment the glucose released by hydrolysis of starch are not capable of fermenting the more diverse mixture of sugars released by hydrolysis of lignocellulosic biomass.
The necessary deconstruction of cellulosic polymers, enzymatic hydrolysis, and saccharification require additional processing procedures to use lignocellulosic biomass ultimately increases the cost of lignocellulose to ethanol conversion when compared to current starch-to-ethanol technologies. Reducing the cost of cellulosic ethanol production poses significant challenges both in scientific advances and technological development.
One barrier is that yeast strains are generally capable of fermenting the hexose sugars, glucose and galactose; however, they do not naturally ferment the pentose sugars, xylose or arabinose without any genetic modification.
Corncobs are commonly used for xylose production, and xylose-extracted corncob residue (X-ER) is an abundant byproduct after industrial processing (Zhang et al., 2011). The X-ER contains a significant amount of cellulose and is a potential feedstock for cellulosic ethanol production. However, ideal processing procedures and economic cellulosic ethanol production from X-ER have not been achieved yet on a large scale (Zhang et al., 2011). More efficient, lower-cost, and consolidated processing procedures are needed.
Simultaneous saccharification and fermentation (SSF) using cellobiose fermenting yeast Brettanoinyces custersii, is described in U.S. Pat. No. 5,100,791, by Spindler, et al. In a simultaneous saccharification fermentation process, saccharification involves the breakdown of cellulose into simpler sugars by a cellulase enzyme. One such sugar is cellobiose, a sugar comprised of two glucose molecules that is subsequently broken down into glucose. The cellulase enzyme will typically have an insufficient amount of β-glucosidase, which is the part of the cellulase enzyme that can breakdown cellobiose into glucose. Cellobiose inhibits the endo- and exo-glucanase enzymes, and this retards the overall ethanol production rate and yield in a simultaneous saccharification fermentation process.
Since the commonly used ethanologenic yeast Saccharomyces cerevisiae is unable to utilize cellobiose, β-glucosidase is added to digest cellobiose into glucose in order to be utilized by the fermentation ethanologenic yeast.
Enzymes are one of the major costs of cellulosic ethanol production (Piccolo and Bezzo, 2009). In addition, efficient enzymatic saccharification requires a higher temperature while microbial growth and fermentation function optimally at a lower temperature. Furthermore, inhibitory compounds such as representative 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-furaldehyde (HMF) are often generated during biomass pretreatments such as commonly used dilute acid pretreatment, that interfere with microbial growth and fermentation (Palmquist and Hahn-Hagerdal, 2000; Liu and Blaschek, 2010). These undesirable elements and redundant processing procedures compromise the efficiency of SSF.
There is a need in the art to develop an ethanologenic yeast strain that is tolerant to both a higher temperature and inhibitors commonly encountered in the SSF. This new yeast produces sufficient native β-glucosidase enzyme activity allowing it to grow on cellobiose as sole source of carbon. Thus, no additional β-glucosidase enzyme needs to be added for cellulosic ethanol conversion from X-ER by SSF. Development of this yeast provides potential consolidated bio-processing means for lower-cost cellulosic ethanol production from industrial byproduct of xylose extracted corncobs and other lignocellulosic biomass materials.